Screening an Na^+/H^+ Antiporter Gene from the Halophiles Colonizing in the Dagong Ancient Brine Well of Zigong City,China

[Objective] This study aimed to screen an Na＋/H＋ antiporter gene from the halophiles colonizing in the Dagong Ancient Brine Well in Zigong City, China, and then analyze the gene structure and properties of the protein encoded by this gene. [Method] Metagenomic DNA libraries of halophiles from the Dagong Ancient Brine Well were used for screening genes with Na＋/H＋ antiporter activity in antiporter-defi- cient E. coil KNabc strain by functional complementation. Then the start codon, stop codon, ORF, -35 region, -10 region and SD sequence of Na~/H＋ antiporter gene, as well as the molecular weight, isoelectric point, hydrophobic region, transmembrane domain, phyletic evolution and salt resistance of protein encoded by the gene were investigated. [Result] A new Na＋/H＋ antiporter gene m-nha was obtained, which ,ren- dered the antiporter-negative mutant E. coil KNabc cells with both the resistance to Na＋ and the ability to grow under alkaline conditions. [Conclusion] The structure and amino acid sequence of M-Nha was different from the previously reported Na＋/H~ antiporters, and the m-nha gene disclosed from the Dagong Ancient Brine Well was identified as a novel Na＋/H＋ antiporter gene. This study was significant not only in helping us understand the salt tolerance of halophiles in ancient brine wells and develop and utilize the genes resource, but also in exploring new salt-tolerant genes.

Characterization and recovery of the halophiles in the fluid inclusions in halite

1 Introduction It is the focus of geology and biology that the creature preserved in the geological history and the organic evolution.The creature preserved in geological history by these things:sedimentary,frozen earth,chrysophoron and evaporation salt.Evaporation salt can preserve the microbe

Isolation and Identity of Halophiles in The Floating Bath Water in Yuncheng

1 Introduction The study of floating bath began in 1914,by Dor.John C.Lily,the expert of medical and neurology.She has done different kinds of floating experiments to study about the origin of consciousness and the relationship with brain.Previous research showed that the effect of floating

Effect of salinization on soil properties and mechanisms beneficial to microorganisms in salinized soil remediation-a review

Salinized soil is an important reserved arable land resource in China.The management and utilization of salinized soil can safeguard the current size of arable land and a stable grain yield.Salt accumulation will lead to the deterioration of soil properties,destroy soil production potential and damage soil ecological functions,which in turn will threaten global water and soil resources and food security,and affect sustainable socio-economic development.Microorganisms are important components of salinized soil.Microbial remediation is an important research tool in improving salinized soil and is key to realizing sustainable development of agriculture and the ecosystem.Knowledge about the impact of salinization on soil properties and measures using microorganisms in remediation of salinized soil has grown over time.However,the mechanisms governing these impacts and the ecological principles for microbial remediation are scarce.Thus,it is imperative to summarize the effects of salinization on soil physical,chemical,and microbial properties,and then review the related mechanisms of halophilic and halotolerant microorganisms in salinized soil remediation via direct and indirect pathways.The stability,persistence,and safety of the microbial remediation effect is also highlighted in this review to further promote the application of microbial remediation in salinized soil.The objective of this review is to provide reference and theoretical support for the improvement and utilization of salinized soil.

An analysis of Great Salt Lake Winogradsky columns

Sergei Winogradsky illuminated revolutionary concepts and produced a tool to visualize complex microbial communities and their metabolisms over time:columns displaying aquatic consortia with variety of niches.We worked with museums in Utah to create Winogradsky columns that would highlight aesthetic properties of the Great Salt Lake(GSL)ecosystem,which has a salinity gradient from the freshwater wetlands to salt saturation.One column,constructed using haloarchaea-rich hypersaline brine and oolitic sand of the lake’s north arm,was enriched with nutrients,and resulted in the desired pink hue over time.After a seven-year maturation period,we examined the microbial taxa present in the water through 16 S/18 S rRNA and Internal Transcribed Spacer(ITS)gene sequencing.A pigment analysis revealed an abundance of bacteriochlorophyll a.The presence of this pigment coupled with the DNA sequencing results,suggest that the haloarchaea that dominate the GSL brine,were not responsible for the pink coloration,but instead Gammaproteobacteria,especially Halorhodospira species.Among the eukaryotes,the lack of phytoplankton and the abundance of fungi were noteworthy observations.These data likely relate to the reduction of oxygen in a non-aerated sealed system over time.Our second exhibit had the goal of educating museum goers about the varying salinities of Great Salt Lake.Here we employed three distinct columns of water and sediment from this salinity gradient.Observations of these columns overtime gave us information about invertebrate communities in addition to the microbial consortia.Both installations taught us about comparing an artificial environment in a museum setting to the natural ecosystem.Taken together,we present the data collected and lessons learned from using Winogradsky columns in public spaces for teaching about an important saline lake.

Characterization of two halophilic adenylate cyclases from Thermobifida halotolerans and Haloactinopolyspora alba

Adenylate cyclase(AC)is the key enzyme that catalyzes the formation of cAMP from ATP.In this study,we discovered two novel class Ⅲ ACs with a halophilic property from Thermobifida halotolerans DSM 44931(ThAC)and Haloactinopolyspora alba DSM 45211(HaAC),respectively.The recombinant ThAC and HaAC were expressed in Escherichia coli with molecular weights of 36.1 and 36.0 kDa respectively.The presence of 2500 and 2200 mmol
L^(-1)1 NaCl significantly enhanced the enzyme activities of ThAC and HaAC,with 22-fold and 7.4-fold higher activities compared to those without NaCl,respectively.Several divalent metal ions were found to activate the recombinant ACs to different extents,and the optimal metal ion was Mg^(2+)for both ThAC and HaAC with concentrations of 80 mmol·L^(-1) and 40 mmol·L^(-1) respectively.Purified ThAC and HaAC had the optimal specific activities((4.59±0.35)×10^(4) and(7.76±0.52)×10^(4) U·mg^(-1))and catalytic efficiency(4.47 and 5.30 L·mmol^(-1)·s^(-1))at 45
℃ and 40
℃ respectively,while the optimum pH of both two recombinant ACs was 10.0.This is the first report of the halophilic Class III ACs,which could make new contributions to explore and study ACs for further associated investigations.

Isolation and characterization of halophilic Bacillus sp.BS3 able to produce pharmacologically important biosurfactants

Objective:To characterize the pharmacological importance of biosurfactants isolated from halophilic Bacillus sp BS3.Methods:Halophilic Bacillus sp.BS3 was isolated from solar salt works,identified by 16S rRNA sequencing and was used for screening their biosurfactant production.Characters of the biosurfactant and their anticancer activity were analyzed and performed in mammary epithelial carcinoma cell at different concentrations.Results:The biosurfactant were characterized by TLC,FTIR and GC-MS analysis and identified as lipopeptide type.GC-MS analysis revealed that,the biosurfactant had various compounds including 13Docosenamide.(Z);Mannosamine,9- and N,N,N',N'-tetramethyl.Surprisingly the antiviral activity was found against shrimp white spot syndrome virus(WSSV) by suppressing the viral replication and significantly raised shrimp survival(P<0.01).Anticancer activity performed in the mammary epithelial carcinoma cell at different concentrations of biosurfactants,among the various concentrations of biosurfactants such as 0.000 25,0.002 5,0.025,0.25 and 2.5 μ g,the 0.25 μ g concentration suppressed the cells significantly(P<0.05) to 24.8%.Conclusions:Based on the findings,the present study concluded that,there is a possibility to develop eco-friendly antimicrobial and anticancer drugs from the extremophilic origin.

A Study of the Characteristics of Microorganisms for Effective Degradation of Marine Oil Spills

Four microorganism strains were isolated from coastal petroleum-polluted soil and sand samples of Bohai Sea oilfield; they were found to degrade marine oil spills effectively. The experimental results show that the degradation efficiency of crude oil with these four strains （XT-4, SZ-1-25, B-4-9, BS-3-12） is 95.97%, 96.01%, 97.99% and 98.99%, respectively, in their optimum conditions. The characteristics of bacterial biodegradation are investigated. The simulation biotreatment of oil-contaminated beach sand, with an initial oil content of 5,664mg/kg-dry-sand, shows that the residual oil content is 2,700 mg/kg-dry-sand and 2,679 mg/kg-dry-sand after 170 days＇ treatment with two bacteria （B-4-9, BS-3- 12）, respectively.

Isolation and characterization of a novel strain of Natrinema containing a bop gene

A novel member of extremely halophilic archaea, strain AJ2, was isolated from Ayakekum Lake located in Altun Mountain National Nature Reserve of Xinjiang Uygur Autonomous Region in China. The strain A J2 requires at least 10% (w/v)NaCl and grows 10% to 30% (optimum at 20%). Phylogenetic analysis based on 16S rDNA sequence comparison revealed that strain A J2 clustered to three Natrinema species with less than 97.7% sequence similarities, suggesting A J2 is a novel member of Natrinema. A bacteriorhodopsin-encoding (bop) gene was subsequently detected in the A J2 genome using the polymerase chain reaction technique. The cloning and sequencing of a 401 base pairs fragment indicated the deduced amino acid sequence of bop from A J2 is different from that reported for bacteriorhodopsins. This is the first reported detection of a bop gene in Natrinema.

Isolation and characterization of halophilic bacteria and archaea from salt ponds in Hangu Saltworks, Tianjin, China

A total of 26 isolates were obtained from solar salt ponds of different salinities(100, 150, 200, and 250) in Hangu Saltworks Co. Ltd., Tianjin, China. Phylogenetic analysis of 16 SrRNA gene sequences indicated that five bacteria genera H alomonas, Salinicoccus, Oceanobacillus, Gracibacillus, and Salimicrobium and one archaea genera H alorubrum were present. The genus H alomonas was predominant with eight strains distributed in a salinity range of 100–200, followed by H alorubrum with six strains in salinity 250. Based on the genus and original sampling salinity, eight bacterial and two archaeal isolates were selected for further morphological, physiological, and biochemical characterization. All of the bacterial strains were moderately halophilic with the optimal salinity for growth being either 50 or 100, while two archaeal strains were extremely halophilic with an optimal growth salinity of 200. Additionally, we put forth strain SM.200-5 as a new candidate S alimicrobium species based on the phylogenic analysis of the 16 SrRNA gene sequence and its biochemical characteristics when compared with known related species.

HOW DO THE RATES OF HALOPHILIC ATTACKS COMPARE WITH THE RATES OF PROTOPHILIC ATTACKS?

The measured relative rates of halophilic and protophilic attacks (k_X/k_H) indicate that the rates of halophilic attacks are comparable in magnitude to those of protophilic attacks (deprotonations).

Characterization of halophilic C_50 carotenoid-producing archaea isolated from solar saltworks in Bohai Bay,China

Halophilic archaea comprise the majority of microorganisms found in hypersaline environments. C50 carotenoids accumulated in archaea cells are considered potential biotechnological products and possess a number of biological functions. Ten red colordes were isolated from brine water in a saltem crystaltizer pond of the Hangu Saltworks, China. 16S rRNA gene sequence analysis showed that the colonies belonged to the extremely halophilic archaea genera Halobacterium and Halorubrum. Two representative strains, Halobacterium strain SP-2 and Halorubrum strain SP-4, were selected for further study on the phenotypic characteristics and effects of salinity and pH on accumulation and composition of pigments in their cells. The archaeal strains were isolated and grown in a culture medium prepared by dissolving yeast extract （10 g/L） and acid-hydrolyzed casein （7.5 g/L） into brine water obtained from a I.ocal salt pond. Their optimum salinity and pH for growth were 250 and 7, respectively, although pigment accumulation （OD490/ mL broth） was highest at pH 8. In addition, at 150-300 salinity, increasing salinity resulted in decreasing pigment accumulation. Analysis of the UV-Vis spectrum, TLC and HLPC chromatograms showed that C50 carotenoid bacterioruberin is the major pigment in both strains.

Biogeographical note on Antarctic microforae: Endemism and cosmopolitanism

This study deals with the biogeography of Antarctic microflora （Antarctica acts as best model to study microbial biogeography） such as cyanobacteria and selected halophiles with special emphasis on Halomonas variabilis and Bacillus licheniformis.Halophiles are known to be resistant not only to salt stress,but also to extreme temperature,pressure,and aridity and they are capable of surviving in harsh environments such as polar regions,deep-sea habitats,and deserts.Many microbes are known to be resistant to hostile environmental conditions,and are capable of surviving in harsh environments.Our group has isolated 444 strains belonging to 28 genera of halophiles from various environments around the world.The 16S rRNA gene sequences revealed that many of the isolated strains from geographically distant habitats having different environmental conditions,were closely related to each other,with some strains possessing 100％ identical sequences.Organisms possessing survival mechanism such as spore formation are usually ubiquitous.The genus Halomonas is represented by potentially endemic strains and the ubiquitous H.variabilis,while spore-forming B.licheniformis showed cosmopolitan distribution.One potentially endemic （moderate endemicity that is regional and/or continental distribution） strain was reported from Syowa station,East Antarctica,and Mario Zucchelli station,West Antarctica,which are geographically separated by 3000 km.Moreover,15 strains having 100％ similarity with B.licheniformis were considered cosmopolitans.The results of this work provide support for the middle-ground model that some microbes have moderate endemicity and others have cosmopolitan distribution.These results will contribute to a greater understanding of microbial biogeography with special emphasis on Antarctica.

Floristic composition of the halophilic and salt-resistant plant population in Hammam-Boughrara (Oran-Algeria)

This phyto-ecological study is on halophilic and salt-resistant vegetation of Oran region. The semiarid and sometimes arid climate has been defined and confirmed from a bioclimatic point of view. The pedological approach used shows a soil with sandy to silty-sandy texture, favoring regression of the vegetation and a halophilic vegetation set up. In this study, we analyze the floristic composition of the northern region of Hammam Boughrara using multiple floristic surveys conducted at three stations along the Tafna wadi. Dominated by Mediterranean and Saharo-Sindian elements, the relatively poor flora (88 species in total) is biologically characterized by a clear dominance of therophytes (>33%) and chamaephytes (>19%) to the detriment of phanerophytes.

Ancient Microorganisms and Carotenoids Preserved in Fluid Inclusions in Halite from Chaka Salt Lake,Western China:Evidence from Micro-observation and in situ Raman Spectroscopy

Trapped ancient microorganisms in halite fluid inclusions are of special interest to the understanding of biology and ecology in salt lake systems.With the integration of petrologic,microthermometric,and Raman spectroscopic analyses,this study utilizes fluid inclusions from Chaka Salt Lake,eastern Qaidam Basin,NW China,to assess the possibility of microorganism-trapping by fluid inclusions.Here,we report that the solid phase of some primary fluid inclusions contains carotenoids,which is interpreted as evidence of Dunaliella algae,and that the coexisting liquid phase comprises SO_(4)^(2-).The homogenization temperatures of single-phase primary fluid inclusions indicate that the precipitation temperature of the Holocene halite in Chaka Salt Lake ranges from 13.5℃ to 36.4℃.This suggests that fluid inclusions in halite are a good medium for trapping and preserving ancient microorganisms and organic matter in salt lakes,and that Raman spectroscopy has good potential to identify halophilic archaea.

Isolation of a Halophilic Bacterium, Bacillus sp. Strain NY-6 for Organic Contaminants Removal in Saline Wastewater on Ship

The objective of this research was to examine if certain strains of Bacillus bacteria, could survive in dry powder products and if so, could the bacteria degrade organic contaminants in saline wastewater on a ship. As part of the study, we isolated 7 domesticated strains named NY1, NY2,..., and NY7, the strain NY6 showed to have the best performance for organic matter degradation and could survive in dry powder more than 3 months. NY6 was identified as Bacillus aerius, based on the morphological and physic-chemical properties. Its optimal growth conditions were as follows： salinity was 2%; temperature was 37℃; pH was in 6.5-7.0; best ratio of C： N： P was 100：5：1. The capability of its dry powder for Chemical Oxygen Demand （COD） removal was 800mg COD/g in synthesized marine wastewater with 2% salinity. The spores in the dry powder were 1.972×108 g-1.

Biodiversity and Screening of Moderately Halophilic Bacteria with Hydrolytic and Antimicrobial Activities from Yuncheng Salt Lake,China

1 Introduction Yuncheng Salt Lake is an old lake with a history of more than 5000 years,which locates in the margins of the central plains of China,with an area of about 130 km.It was an important resource of salt for human consumption from early times.Major chemicals in Yuncheng Salt Lake contain Na Cl,Na2SO4 and Mg SO4(Gao et al.2007).In recent years,some halophilic bacteria and archaea were

Isolation and in-situ bioremediation of halophile oil degradation bacteria

Crude oil exploration has resulted in alarming pollution. It is thereforenecessary to isolate native bacterial strains for contaminated crude oil bioremediation.In this study, three bacteria strains were isolated from the Dagang Oil Field, Tianjing,China, which belonged to the genera Pseudomonas sp. and Bacillus sp., as determinedby 16S rRNA genera sequence similarity analysis. The optimum degradation conditionsof the three bacteria were studied by single factor experiment, and they were 30 °C, 7.2pH, and 3% salinity, 35 °C, 7.5 pH, and 3% salinity, and 37 °C, 7.5 pH, and 5% salinity,respectively. The results showed that the oil degradation rate was the best when theproportion of the different strains was 2:1:2. Moreover, the most effective treatmentconditions were found by orthogonal test to be 10% inoculum, 0.2% oil content, 7.0 pH,3:1 N:P, 3.5% salinity, and 35 °C, the oil degradation rate reached 71.03% under thiscondition.

Halophilic Vibrios in the waters of Xiamen Harbor and Daya Bay

The distribution featires, spotes composition and seasonal variation of halophilic Vibrios in 7 stations ofXiamen Harbor and 9 stations of the Daya Bay were studied. The counts of Vibrios were analyzed with the most probable number(MPN) technique. The media TCBS was used to isolate Vibrios, and API20E system employed to identifythe Vibrios. The results reveals that the density Of Vibrios in the Daya Bay ranged from 30. 0 x 105 to 2. 4 x 105 celldin-3 and the average density was 6. 61 x 103 cell·din-3, and that in Xiamen Harbor was 2. 3 x 102-2. 4 x 105 cen·din-3 and 7. 8 x 103 cell·din-3 on the average. The number of Vibrios varied seasonally with the water temperature,and was heher in the summer than in the autumn. The Vibrio species in the two bays mainly included Vibrioalginolyticus, V. parahaemolytic-us, V. fluvialis, V. vulnificus, V. mimices and V. metshnikovii and V.alginolyticus was the predominant spotes.

Effects of Sea Salts on Induction of Cell Proliferation in Liquid Cultures of Mangrove Plants, Sonneratia caseolaris and S. alba

The effects of five salt ingredients of sea water, KCl, NaCl, CaCl2, MgCl2 and MgSO4, on induction of cell prolif-eration in Sonneratia caseolaris were investigated. Proliferation was examined in tissue explants derived from such as leaves, cotyledons, and hypocotyls using a small-scale liquid culture method. Addition of 12.5-25 mM of MgCl2 was unique in stimulating cell proliferation in all tissues of S. caseolaris. Otherwise, different effects of salts were observed among the three tissues. In hypocotyl culture, 25-50 mM of NaCl and CaCl2 stimulated cell divisions. Tolerance to 100 mM of MgSO4 was observed in leaves. Three osmotically active compounds commonly used in tissue culture, sorbitol, mannitol and glycinebetaine, were also tested to assess the importance of osmotic effects on cell proliferation. No significant stimulation by these was observed over a wide range of concentrations. Data were compared with those of cotyledon cultures of another mangrove, S. alba, which exhibits no stimulation by MgCl2, stimulation by KCl and tolerance to NaCl. Mechanisms for adaptation of mangrove plants to various and high salts were discussed by comparing the differences in reaction to salts in cultures of two Sonneratia mangrove species of the same genera growing different salt environment.